Impact of maternal protein supplementation during mid or late gestation on skeletal muscle energy metabolism of beef calves / Impacto da suplementação proteica materna durante o terço médio ou terço final da gestação sobre o metabolismo energético do tecido muscular de bezerros de corte

ABSTRACT: This study evaluated the effect of maternal protein supplementation during mid or late gestation on energy metabolism of the skeletal muscle of beef calves. Sixteen pregnant cows were divided into 3 groups: CTRL (not supplemented); MID (supplemented from 30 to 180 days of gestation); and LATE (supplemented from 181 to 281 days of gestation). The supplement contained 30% crude protein. Thirty days after birth, blood and muscle samples of the calves were collected for analyses of gene expression, proteins, and metabolites. No differences (P ≥ 0.15) in birth weight, performance at weaning, or muscle expression of the genes evaluated (P ≥ 0.21) were observed. Calves born to CTRL cows had a lower ratio (P = 0.03) of p-AMPK/AMPK protein in the skeletal muscle. Calves born to MID cows had lower (P = 0.04) glucose concentration than those born to LATE cows. Changes in p-AMPK/AMPK protein, indicated a possible metabolic inflexibility in the skeletal muscle of calves born to CTRL cows. These results indicated that lack of protein supplementation in pregnant cows alter the energy metabolism of their calves and reflect in a metabolic inflexibility.

RESUMO: O objetivo deste estudo foi avaliar o efeito da suplementação proteica materna sobre o metabolismo energético do músculo esquelético de bezerros de corte. Dezesseis matrizes gestantes foram divididas em três grupos: CONTROLE (não suplementado); MÉDIO (suplementados entre 30 e 180 dias de gestação); e FINAL (suplementado entre 181 e 281 dias de gestação). O suplemento continha 30% de proteína bruta e foi fornecido em quantidades totais iguais aos tratamentos. Trinta dias após o nascimento, amostras de sangue e músculo dos bezerros foram coletadas para análises de expressão gênica, abundância de proteínas e metabólitos. Não foram observadas diferenças (P ≥ 0,15) no peso ao nascimento ou parâmetros de desempenho ao desmame, bem como na expressão dos genes avaliados (P ≥ 0,21). Os bezerros nascidos de matrizes do tratamento CONTROLE apresentaram menor proporção (P = 0,03) de proteína p-AMPK/AMPK no músculo esquelético. Os bezerros nascidos de matrizes do tratamento MÉDIO apresentaram concentração de glicose menor (P = 0,04) do que aqueles nascidos de matrizes do tratamento FINAL. Os resultados observados indicam que a ausência de suplementação proteica em matrizes gestantes pode alterar o metabolismo energético da progênie e refletir em uma inflexibilidade metabólica, a qual pode ocasionar limitações quanto à eficiência energética do tecido muscular esquelético e consequentemente, limitar o desempenho da progênie ao longo da fase pós-natal.

Recombinant expression and antigenicity of two peptide families of neurotoxins from Androctonus sp

Background: Scorpion neurotoxins such as those that modify the mammalian voltagegated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes ­ one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 ­ and another non-native toxin ­ named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions ­ were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.(AU)

cDNA Cloning of a Bovine Insulin-like growth factor-1 from Egyptian Buffalos and Expression of its Recombinant Protein in Escherichia coli / Clonagem de cDNA de um fator-1 de crescimento semelhante à insulina bovina de búfalos egípcios e expressão de sua proteína recombinante em Escherichia coli

Insulin-like growth factor-1 (IGF-1) is regarded as a crucial clinically significant therapeutic agent against several pathological conditions. Recently, recombinant DNA (rDNA) technology has enabled the production of many drugs of rDNA-origin including IGF-1. Securing a readily available supply of IGF-1 is invaluable to clinical research and biotechnological domains. In this work, the cloning of a full-length bovine IGF-1 cDNA and the successful expression of its cognate recombinant IGF-1 protein is reported. Single-strand cDNA was prepared from liver tissues, through the specific reverse transcription (RT) of IGF-1 mRNA. Subsequently, a PCR amplicon of ~543bp was successfully amplified. Recombinant pTARGET™ vector harboring IGF-1 insert was successfully cloned into competent E. coli JM109 cells. SDS-PAGE analysis revealed that the recombinant IGF-1 has been expressed at the expected size of 7.6kDa. The outcome provides a robust basis for transecting the recombinant pTARGETTM vector, harboring the IGF-1 cDNA insert, into mammalian cells. Optimal initial glucose concentration was found to be 10g/l with corresponding protein concentration of 6.2g/l. The proliferative biological activity crude recombinant IGF-1 protein was verified on HeLa cell lines. This is envisaged to facilitate large-scale production of recombinant IGF-1 protein, thereby enabling thorough investigation of its clinical and pharmaceutical effects.(AU)

O fator de crescimento semelhante à insulina-1 (IGF-1) é considerado um agente terapêutico clinicamente significativo contra várias condições patológicas. Recentemente, a tecnologia de DNA recombinante (rDNA) permitiu a produção de muitos medicamentos de origem rDNA, incluindo o IGF-1. Garantir um suprimento prontamente disponível de IGF-1 é inestimável para pesquisas clínicas e domínios biotecnológicos. Neste trabalho, relata-se a clonagem de um cDNA de IGF-1 bovino de comprimento total e a expressão bem-sucedida de sua proteína IGF-1 recombinante cognata. O cDNA de cadeia simples foi preparado a partir de tecidos do fígado, por meio da transcrição reversa específica (RT) do mRNA de IGF-1. Posteriormente, um amplificador de PCR de ~ 543pb foi amplificado com sucesso. O vetor pTARGET™ recombinante contendo a inserção de IGF-1 foi clonado com sucesso em células competentes E. coli JM109. A análise por SDS-PAGE revelou que o IGF-1 recombinante foi expresso no tamanho esperado de 7,6kDa. O resultado fornece uma base robusta para a transferência do vetor pTARGETTMTM recombinante, abrigando a inserção de cDNA de IGF-1 em células de mamíferos. Verificou-se que a concentração inicial ideal de glicose é 10g/L, com a concentração de proteína correspondente de 6,2g/L. A proteína IGF-1 recombinante bruta de atividade biológica proliferativa foi verificada nas linhas celulares HeLa. É previsto que isso facilite a produção da proteína IGF-1 recombinante em larga escala, permitindo, assim, uma investigação completa dos seus efeitos clínicos e farmacêuticos.(AU)

cDNA Cloning of a Bovine Insulin-like growth factor-1 from Egyptian Buffalos and Expression of its Recombinant Protein in Escherichia coli / Clonagem de cDNA de um fator-1 de crescimento semelhante à insulina bovina de búfalos egípcios e expressão de sua proteína recombinante em Escherichia coli

Insulin-like growth factor-1 (IGF-1) is regarded as a crucial clinically significant therapeutic agent against several pathological conditions. Recently, recombinant DNA (rDNA) technology has enabled the production of many drugs of rDNA-origin including IGF-1. Securing a readily available supply of IGF-1 is invaluable to clinical research and biotechnological domains. In this work, the cloning of a full-length bovine IGF-1 cDNA and the successful expression of its cognate recombinant IGF-1 protein is reported. Single-strand cDNA was prepared from liver tissues, through the specific reverse transcription (RT) of IGF-1 mRNA. Subsequently, a PCR amplicon of ~543bp was successfully amplified. Recombinant pTARGET™ vector harboring IGF-1 insert was successfully cloned into competent E. coli JM109 cells. SDS-PAGE analysis revealed that the recombinant IGF-1 has been expressed at the expected size of 7.6kDa. The outcome provides a robust basis for transecting the recombinant pTARGETTM vector, harboring the IGF-1 cDNA insert, into mammalian cells. Optimal initial glucose concentration was found to be 10g/l with corresponding protein concentration of 6.2g/l. The proliferative biological activity crude recombinant IGF-1 protein was verified on HeLa cell lines. This is envisaged to facilitate large-scale production of recombinant IGF-1 protein, thereby enabling thorough investigation of its clinical and pharmaceutical effects.(AU)

O fator de crescimento semelhante à insulina-1 (IGF-1) é considerado um agente terapêutico clinicamente significativo contra várias condições patológicas. Recentemente, a tecnologia de DNA recombinante (rDNA) permitiu a produção de muitos medicamentos de origem rDNA, incluindo o IGF-1. Garantir um suprimento prontamente disponível de IGF-1 é inestimável para pesquisas clínicas e domínios biotecnológicos. Neste trabalho, relata-se a clonagem de um cDNA de IGF-1 bovino de comprimento total e a expressão bem-sucedida de sua proteína IGF-1 recombinante cognata. O cDNA de cadeia simples foi preparado a partir de tecidos do fígado, por meio da transcrição reversa específica (RT) do mRNA de IGF-1. Posteriormente, um amplificador de PCR de ~ 543pb foi amplificado com sucesso. O vetor pTARGET™ recombinante contendo a inserção de IGF-1 foi clonado com sucesso em células competentes E. coli JM109. A análise por SDS-PAGE revelou que o IGF-1 recombinante foi expresso no tamanho esperado de 7,6kDa. O resultado fornece uma base robusta para a transferência do vetor pTARGETTMTM recombinante, abrigando a inserção de cDNA de IGF-1 em células de mamíferos. Verificou-se que a concentração inicial ideal de glicose é 10g/L, com a concentração de proteína correspondente de 6,2g/L. A proteína IGF-1 recombinante bruta de atividade biológica proliferativa foi verificada nas linhas celulares HeLa. É previsto que isso facilite a produção da proteína IGF-1 recombinante em larga escala, permitindo, assim, uma investigação completa dos seus efeitos clínicos e farmacêuticos.(AU)

Heterologous expression and mutagenesis of recombinant Vespa affinis hyaluronidase protein (rVesA2)

Background:Crude venom of the banded tiger waspVespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors.Methods:The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed.Results:V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)7 composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4.Conclusion:The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity.(AU)

Heterologous expression and mutagenesis of recombinant Vespa affinis hyaluronidase protein (rVesA2)

Background:Crude venom of the banded tiger waspVespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors.Methods:The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed.Results:V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)7 composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4.Conclusion:The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity.

Taurine Reduces Heat Stress by Regulating the Expression of Heat Shock Proteins in Broilers Exposed to Chronic Heat

ABSTRACT The aim of the current study was to investigate the effects of taurine on heat stress by evaluating them RNA and protein expressions of HSP90, 70 and 60in Ross broilers. Birds were distributed in a control group (CO) reared at 24ºC, a heat-stress group (HS) maintained at 34°C (weeks 3-5) and not supplemented with taurine, and a heat-stress group (HST) maintained at 34°C (weeks 3-5) and supplemented with 0.1% taurine from 2 weeks of age. The final body weight was significantly higher in the HST group than in the HS group (p 0.05). The mRNA expression of all three genes in the liver and of HSP90 in the muscle were significantly lower in the HST than in the HS group (p 0.05). In the liver, the expression of HSP70 and HSP60 proteins was significantly higher in the HS group compared with the CO and HST groups (p 0.05), while HSP90 expression was not different (p>0.05). In the muscle, HSP70 expression was significantly lower in the HST group than in the CO and HS groups and HSP60 expression was dramatically decreased in HS group, whereas no differences in HSP90 expression were detected among groups. In conclusion, the broilers exposed to heat stress and supplemented with taurine showed lower expressions of heat shock genes and proteins both in the liver and muscle tissues, indicating that taurine supplementation improved broiler thermo-tolerance.

Taurine Reduces Heat Stress by Regulating the Expression of Heat Shock Proteins in Broilers Exposed to Chronic Heat

The aim of the current study was to investigate the effects of taurine on heat stress by evaluating them RNA and protein expressions of HSP90, 70 and 60in Ross broilers. Birds were distributed in a control group (CO) reared at 24ºC, a heat-stress group (HS) maintained at 34°C (weeks 3-5) and not supplemented with taurine, and a heat-stress group (HST) maintained at 34°C (weeks 3-5) and supplemented with 0.1% taurine from 2 weeks of age. The final body weight was significantly higher in the HST group than in the HS group (p 0.05). The mRNA expression of all three genes in the liver and of HSP90 in the muscle were significantly lower in the HST than in the HS group (p 0.05). In the liver, the expression of HSP70 and HSP60 proteins was significantly higher in the HS group compared with the CO and HST groups (p 0.05), while HSP90 expression was not different (p>0.05). In the muscle, HSP70 expression was significantly lower in the HST group than in the CO and HS groups and HSP60 expression was dramatically decreased in HS group, whereas no differences in HSP90 expression were detected among groups. In conclusion, the broilers exposed to heat stress and supplemented with taurine showed lower expressions of heat shock genes and proteins both in the liver and muscle tissues, indicating that taurine supplementation improved broiler thermo-tolerance.(AU)

Taurine Reduces Heat Stress by Regulating the Expression of Heat Shock Proteins in Broilers Exposed to Chronic Heat

The aim of the current study was to investigate the effects of taurine on heat stress by evaluating them RNA and protein expressions of HSP90, 70 and 60in Ross broilers. Birds were distributed in a control group (CO) reared at 24ºC, a heat-stress group (HS) maintained at 34°C (weeks 3-5) and not supplemented with taurine, and a heat-stress group (HST) maintained at 34°C (weeks 3-5) and supplemented with 0.1% taurine from 2 weeks of age. The final body weight was significantly higher in the HST group than in the HS group (p 0.05). The mRNA expression of all three genes in the liver and of HSP90 in the muscle were significantly lower in the HST than in the HS group (p 0.05). In the liver, the expression of HSP70 and HSP60 proteins was significantly higher in the HS group compared with the CO and HST groups (p 0.05), while HSP90 expression was not different (p>0.05). In the muscle, HSP70 expression was significantly lower in the HST group than in the CO and HS groups and HSP60 expression was dramatically decreased in HS group, whereas no differences in HSP90 expression were detected among groups. In conclusion, the broilers exposed to heat stress and supplemented with taurine showed lower expressions of heat shock genes and proteins both in the liver and muscle tissues, indicating that taurine supplementation improved broiler thermo-tolerance.

A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

Background:The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.Methods:The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.Results:Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.Conclusions:The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.(AU)

A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

Background: The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. Methods: The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Results: Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. Conclusions: The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.(AU)

A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide

Background: The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. Methods: The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Results: Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. Conclusions: The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.

Construction of adenoviral vectors expressing F and G glycoproteins of human respiratory syncytial virus (HRSV)

Human Respiratory Syncytial Virus (HRSV) was first characterized in 1957 and has since been recognized as the most common viral cause of severe respiratory tract infection in young infants worldwide. Despite many years of research there is still no effective treatment or any immediate prospect of a vaccine. The HRSV genome is composed of single stranded negative sense RNA and the virion consists of a nucleocapsid packaged within a lipid envelope. The envelope contains spike-like projections, each being a homo-oligomer of one of three transmembrane viral envelope proteins: the attachment protein G, the fusion protein F involved in viral penetration and the small hydrofobic protein SH. The aim of this work was to construct two recombinant replication-defective adenoviruses carrying separately F and G genes from HRSV. This system was chosen because adenovirus delivers genes into target cells with high efficiency in a variety of cell lines and can be used in vitro and in vivo. In order to obtain the recombinant viruses, we did RT-PCR of RNA extracted from the HRSV A2 strain, the genes F and G were cloned in to pAdeno-X vectors. pAdeno-F and pAdeno-G were transfected in HEK-293 cells for the production of recombinant viruses, that expressed efficiently these two proteins and provide us the means for doing functional assays and immunization tests.

O Vírus Sincicial Respiratório Humano (HRSV) foi isolado e caracterizado pela primeira vez em 1957 e é considerado como o patógeno viral mais freqüente do trato respiratório de bebês e crianças. Apesar de muitos anos de pesquisa, não há ainda um tratamento específico ou uma vacina licenciada. Seu genoma é composto por uma fita simples de RNA polaridade negativa e o vírion consiste em um nucleocapsídeo empacotado por um envelope lipídico. O envelope contém projeções, chamadas espículas, constituídas de homoligômeros de uma das 3 glicoproteínas de membrana: a proteína de ligação G ("attachment"), a proteína de fusão F ("fusion") e a proteína SH ("small hydrofobic"). O objetivo deste trabalho foi construir dois adenovirus recombinantes defectivos em replicação expressando separadamente os genes F e G do HRSV. Este sistema foi escolhido porque os vetores adenovirais possuem a capacidade de inserir genes em uma grande variedade de linhagens celulares in vitro e in vivo. Para obtenção destes vetores adenovirais, um RT-PCR de RNA extraído do protótipo A2 de HRSV foi feito e os genes F e G clonados em vetores pAdeno-X. pAdeno-F e pAdeno-G foram transfectados em células HEK-293 para a produção do vírus recombinante, que expressaram corretamente essas duas proteínas constituem-se ferramentas para imunização e estudos funcionais.

Construction of adenoviral vectors expressing F and G glycoproteins of human respiratory syncytial virus (HRSV)

Human Respiratory Syncytial Virus (HRSV) was first characterized in 1957 and has since been recognized as the most common viral cause of severe respiratory tract infection in young infants worldwide. Despite many years of research there is still no effective treatment or any immediate prospect of a vaccine. The HRSV genome is composed of single stranded negative sense RNA and the virion consists of a nucleocapsid packaged within a lipid envelope. The envelope contains spike-like projections, each being a homo-oligomer of one of three transmembrane viral envelope proteins: the attachment protein G, the fusion protein F involved in viral penetration and the small hydrofobic protein SH. The aim of this work was to construct two recombinant replication-defective adenoviruses carrying separately F and G genes from HRSV. This system was chosen because adenovirus delivers genes into target cells with high efficiency in a variety of cell lines and can be used in vitro and in vivo. In order to obtain the recombinant viruses, we did RT-PCR of RNA extracted from the HRSV A2 strain, the genes F and G were cloned in to pAdeno-X vectors. pAdeno-F and pAdeno-G were transfected in HEK-293 cells for the production of recombinant viruses, that expressed efficiently these two proteins and provide us the means for doing functional assays and immunization tests.

O Vírus Sincicial Respiratório Humano (HRSV) foi isolado e caracterizado pela primeira vez em 1957 e é considerado como o patógeno viral mais freqüente do trato respiratório de bebês e crianças. Apesar de muitos anos de pesquisa, não há ainda um tratamento específico ou uma vacina licenciada. Seu genoma é composto por uma fita simples de RNA polaridade negativa e o vírion consiste em um nucleocapsídeo empacotado por um envelope lipídico. O envelope contém projeções, chamadas espículas, constituídas de homoligômeros de uma das 3 glicoproteínas de membrana: a proteína de ligação G ("attachment"), a proteína de fusão F ("fusion") e a proteína SH ("small hydrofobic"). O objetivo deste trabalho foi construir dois adenovirus recombinantes defectivos em replicação expressando separadamente os genes F e G do HRSV. Este sistema foi escolhido porque os vetores adenovirais possuem a capacidade de inserir genes em uma grande variedade de linhagens celulares in vitro e in vivo. Para obtenção destes vetores adenovirais, um RT-PCR de RNA extraído do protótipo A2 de HRSV foi feito e os genes F e G clonados em vetores pAdeno-X. pAdeno-F e pAdeno-G foram transfectados em células HEK-293 para a produção do vírus recombinante, que expressaram corretamente essas duas proteínas constituem-se ferramentas para imunização e estudos funcionais.